Protocol for the cloning of pSHAG-MAGIC2 shRNAs.
Comments on the use of shRNAs in mammals.
RNA interference has now gained popularity in a variety of biological systems as
a methodology of choice for knocking down gene expression (reviewed in Hannon,
2002). In mammals, the demonstration of gene silencing using dsRNA products <30bp
in length in well worn in vitro cell systems has placed RNAi at the fore front of gene
manipulation techniques in somatic cells. Three types of dsRNA triggers are now
commonly used to evoke RNAi in mammalian cells: (1) in vitro, RNAseIII “processed”
long dsRNA; (2) chemically or in vitro synthesized small interfering RNAs (siRNA); and
(3) short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters
(reviewed in Paddison and Hannon, 2002).
Our lab has chosen the latter approach for several reasons: first, the considerable
cost of chemically synthesized siRNAs; second, the possibility of enforceable and stable
expression of shRNAs; third applications of expression constructs in primary cell types
(eg, using retroviruses) and in whole organisms (eg, mouse). Our method is to drive
expression of shRNAs by placing them behind the human RNA polymerase III U6
promoter. This expression system has now been demonstrated effective both in vitro
(Paddison et al, 2002; Paddison and Hannon, 2002; Hemann et al, 2003) and in vivo
transiently in mouse (McCaffery et al, 2002), stably during hematopeiosis (Hemann,
2003) and stably in the generation of transgenic mice (Carmell et al, 2003).
For small-scale applications using shRNAs (eg, knocking out a handful of genes),
we strongly recommend constructing 3-6 shRNAs per gene and carrying out a validation
step before doing the actual biological experiment. For example, we often will
transiently transfect shRNA plasmids into any commonly used cell line which also
expresses the gene of interest and assay efficacy by Western, Northern or RT-PCR.
Alternately, the target gene can be introduced transiently along with the shRNA and
assayed in a cell type lacking endogenous expression. Western blots or RT-PCR are
good predictors of efficacy in these assays. We find a direct correlation between whether
a shRNA works well in transient assays and when expressed stably (eg from a retrovirus).
In fact, we have been able to build “epi-allelic” series by this method - a series of
shRNAs with different efficacies which then give rise to phenotypes of a corresponding
severity. The correlation of knockdown and phenotype appears to hold true from
transient and stable experiments in vitro to stable expression in vivo (Hemann et al,
2003).
Concerning in vivo experiments in mouse, we have recently demonstrated that
shRNAs can be used to make transgenic mice, and that the silencing effect is
transmittable to the next generation (Carmell et al, 2003). The important finding here is
that mammalian development does not seem to present a barrier to the use of RNAi as a
genetic tool (eg RNAi is likely “on” most of the time in most tissues). We’ve had the
most success in making transgenics by first stably expressing shRNAs in ES cells, and
validating that shRNA expressing clones do in fact silence the intended target gene
before making chimeras. Again, the notion to stress is pre-validation. If your gene of
interest is not expressed in ES cells you can always try to express it transiently the hairpin
expressing clones. We are experimenting with both lentivirus and MSCV-based systems
for our ES cell work and foresee no barrier to implementing their use.
One of the interesting differences of RNAi in mammals is the apparent lack of
amplification and transport of the silencing trigger. As opposed to C. elegans or plants,
mammalian RNAi appears to produce a transient, cell autonomous targeting effect.
Triggers of RNAi have half-lives and, at least for the time being, must be continuously
feed to the RNAi machinery to maintain silencing. Thus, stable RNAi requires stable
expression of the dsRNA trigger. [[Obviously, this is subject to change if transcriptional
gene silencing is available in mammals.]] For this purpose, we and others have
developed retroviral expression constructs for stable expression of shRNAs (Paddison &
Hannon, 2002; Hemann et al, 2003).
For retroviral vectors in general, we have had the most success putting the shRNA
expression cassette between the 5’ LTR and the drug selection marker. For example, the
configuration of our MSCV-puro construct is shown below.
LTR

U6 pro
shRNA
PGK
PUROR
LTR
We have carried out expression analysis of shRNA cassettes from the LTR and MCS
positions (shown above) and have found that the MCS configuration seems to generally
K cassettes. The use of selfwork better when comparing the same shRNAs expression
inactivating virus appeared to make no difference on silencing in vitro and in vivo in the
MSCV system (however, we are still testing this).
In order to produce retroviruses from packaging cell lines, we find that no
modifications are required to existing protocols for Lenti- and MSCV-based viral
production.
Introduction to pSHAG-MAGIC2
We have recently adopted a new shRNA-based method for evoking RNAi in
mammals. After months of comparisons, we have replaced our old design (a simple 29bp
hairpin with a 4nt loop) with a design based on the Human miR30 microRNA (miRNA).
We decided to change strategies for several reasons. First, adding the miR30 loop and
125nt of miR30 flanking sequence on either side of the hairpin resulted in >10-fold
increase in Drosha and Dicer processing of the expressed hairpins when compared to the
old designs. Increased Drosha and Dicer processing, translates into greater siRNA
production and greater potency for expressed haripins. Second, by using the miR30
designs we can incorporate “rule-based” designs for target sequence selection similar to
those recently proposed by Phil Zamore’s group for endogenously expressed miRNAs.
One such rule is the unstablizing the 5’ end of antisense strand which results in strand
specific incorporation of miRNAs into RISC. Lastly, the miR30 design offers more
flexibility in applications of gene silencing, as they can also be expressed from RNA
polymerase II promoters (eg, CMV) or even arrays of different hairpins in poly-cistronic
transcripts.
Our pSHAG-MAGIC2 cloning vector is roughly equivalent to pSHAG-MAGIC1
(see Paddison et al., 2004) with a few notable exceptions. First is that the cloning
strategy has been changed. We previously had used “PCR-SHAG” to clone hairpins by
adding the entire hairpin onto the end of a PCR primer. We now use a single oligo,
which contains the hairpin and common 5’ and 3’ ends, as a PCR template. That is, we
actually PCR amplify the oligo itself using universal primers which contain XhoI (5’
primer) and EcoRI (3’ primer). These PCR fragments are then cloned into the hairpin
cloning site of pSHAG-MAGIC2. As before, we are expressing the mir30-styled
hairpins from the human U6 promoter.
The configuration of pSHAG-MAGIC2 is shown below (for more detailed map
see attached figure.) The 5’ and 3’ flanks are derived from 125 bases of sequencing
surrounding the Human miR30 miRNA.
LTR

U6 pro
5’flank-shRNA-3’flank
PUROR
PGK
LTR
LTR
 U6 pro
K
The final, pre-processed structure of the miR30-styled shRNAs is shown below.
A
A
----A
UG GCG GCUGCUGGUGCCAACCCUAUUUA
GUG A
AU CGU CGACGACCACGGUUGGGAUAAAU
CAC G
C
G
GUAGA
C
Oligo design
Sense 22mer (Black)
A
A
-----
A
miR30 loop
UG GCG GCUGCUGGUGCCAACCCUAUUUA
GUG A
Structure
AU CGU CGACGACCACGGUUGGGAUAAAU
CAC G (yellow)
C
G
GUAGA
C
Anti-sense/target sequence 22mer (blue)
Common mir30 contexts regions shown in red
The mir30-styled shRNA is synthesized as a single stranded DNA oligo with common
ends corresponding to part of the endogenous mir30 miRNA flanking sequence. These
common sequences are used to prime a PCR reaction, whereby the entire mir30-styled
shRNA is amplified to produce a clonable PCR product.
Template oligo  single strand 97nt “mir30-like” DNA oligo
TGCTGTTGACAGTGAGCGAGCTGCTGGTGCCAACCCTATTTAGTGAAGCCAC
AGATGTAAATAGGGTTGGCACCAGCAGCGTGCCTACTGCCTCGGA
5'miR30PCRxhoIF
shRN
cagaaggctcgagaaggtatattgctgttgacagtgagcg
3'miR30PCREcoRIF
ctaaagtagccccttgaattccgaggcagtaggca
PCR amplification
For 100uL reactions.
10uL
5uL
5uL
2uL
1uL
1uL
1uL
1uL
74uL
10x reaction buffer
DMSO
GC-melt*
dNTPs (10mM stock)
5’ primer (50uM stock)
3’ primer (50uM stock)
Template oligo (100ng/uL)
Vent DNA polymerase (NEB)
H2O
*Unfortunately, the GC-melt PCR reagent is only available from Clontech in the
Advantage-GC PCR kit, not as an individual reagent. You can use this kit, however, for
individual mir30 –shRNA clonings with reasonable success. For a number of reasons,
we have a very strong suspicion that the GC-melt reagent is simply betaine, but we have
not yet validated the use of betaine itself in the PCR reactions.
PCR program
94o
:30
94o
:30
54o
:30
75o 1:00
75o 10:00
4o forever
25 cycles
Cloning
The PCR should product a single band of bp. I use one 100uL PCR reaction per
cloning. Given the small size of the PCR product, I do not recommend column-based
clean up kits. We clean up the PCR reaction by (1) phenol: chloroform extraction
followed by a single chloroform extraction and (2) ethanol precipitation with 1:10
volume of 3M NaAc (pH=4.8 ) [adding glycogen as a pellet marker is advised] and (3)
washing in 70% ethanol. Resuspend the pellet is 10mM Tris or H2O and proceed with
EcoRI & XhoI digestion. I usually cut for 1.5hrs.
Gel purify the fragment using 2% high temp melt agarose. I use QiaxII gel
purificaiton kit (ie the glass bead kit). After purification resuspend in 20uL of 10mM
Tris, pH 7-8. Metaphor Agarose gives excellent resolution for these separations but is
not essential. For vector preparation, I EcoRI/XhoI cut 2ug of pSM2, gel purify, and
resuspend in 20uL of 10mM Tris, pH 7-8.
For ligations I’ve had the best luck adding 3-5uL of cut PCR product and 1uL of
vector in a 20uL ligation reaction. I use NEB high concentration T4 DNA ligase. Leave
the ligation for 10min to 2hrs (depending on your need) at RT and transform 5-10uL of
the ligation.
Important: ligations must be transformed into PIR1 competent bacteria
[Invitrogen and also available from S. Elledge]. The pSM2 plasmid harbors a
conditional bacterial origin of replication which requires the expression of the “pir1”
gene to be rendered functional. Recombinants should be selected using BOTH
chloramphenicol (25ug/ml) and kanamycin (25ug/ml). Selecting with only one or the
other will result in higher than expected background.
Sequencing
As a matter of practice with shRNAs, we only use sequence verified shRNAs for
biological experiments. We find that oligo synthesis can very quite a lot. So sequence
verify at least six CmR/KanR bacterial clones per oligo cloned when cloning individual
shRNAs or at least three-fold coverage when sequencing complex cloning pools (ie 310,000 shRNAs).
U6 sequencing primer(-42 from start of transcription):
GTAACTTGAAAGTATTTCG
The sequence of pSHAG-MAGIC2
5’ MSCV LTR
Packaging signal
U6 promoter + 27nt leader sequence…2003-2515
5’ mir30 context…2515-2634
Cloning site [XhoI-EcoRI] 2634-2646
3’ mir30 context…2646-2760
U6 terminator…2760-2764
Barcode cloning site [MfeI-MluI]… 2765-2799
Chloramphenicol resistance gene…2809-3763
PGK-PURO
3’ MSCV-“SIN”-LTR
CTTCCCAACCTTACCAGAGGGCGCCCCAGCTGTCCGAAATATTATAAATTATCGCACAC
ATAAAAACCATGCTGTTGGTGTGTCTATTAAATCGGCAACTGTTGGGAAGGGCGATCGG
TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTA
AGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCGCAAGGAAT
GGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACC
CACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGA
TGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATG
CGTCCGGCGTAGAGGCGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGC
TCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAA
AATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGG
TTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGA
ATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGG
ATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGAT
GCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGG
ACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGT
TCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGC
CAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTG
TTTGCATCCGAATCGTGGACTCGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTGACT
GCCCACCTCGGGGGTCTTTCATTTGGAGGTTCCACCGAGATTTGGAGACCCCTGCCCAG
GGACCACCGACCCCCCCGCCGGGAGGTAAGCTGGCCAGCGGTCGTTTCGTGTCTGTCTC
TGTCTTTGTGCGTGTTTGTGCCGGCATCTAATGTTTGCGCCTGCGTCTGTACTAGTTAG
CTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCTGAACACCCGGC
CGCAACCCTGGGAGACGTCCCAGGGACTTTGGGGGCCGTTTTTGTGGCCCGACCTGAGG
AAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATGTGGTTCTGGTAGGAGACGAGA
ACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGAACCGAAGCCGC
GCGTCTTGTCTGCTGCAGCGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTT
TCTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAG
GTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGA
CGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGA
CGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCC
CGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGAC
CCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATC
CGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATC
CAGCCCTCACTCCTTCTCTAGGCGCCGGAATTAGATCTCTCGATAATAGGGGACCGGAT
CCCCCCGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGCCCCTCGCTCCAAAAA
TGCTTTCGCGTCTCGCAGACACTGCTCGGTAGTTTCGGGGATCAGCGTTTGAGTAAGAG
CCCGCGTCTGAACCCTCCGCGCCGCCCCGGCCCAGTGGAAAGACGCGCAGGCAAAACGC
ACCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGG
CCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAA
TTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAG
TAATAATTTCTTGGGTAGTTTGCAGTTTTTAAAATTATGTTTTAAAATGGACTATCATA
TGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGA
CGAAACACCGTGCTCGCTTCGGCAGCACATATACTAGTCGACTAGGGATAACAGGGTAA
TTGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACT
TGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTCGAGCAACCAGAATTCAAGG
GGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGA
TACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTCAATTGGAA
GACTAATGCGTTTAAACACGCGGCGACGCGTTCGACCGAATAAAACCTGTGACGGAAGA
TCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCA
ACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAAT
GAAATAAGATCACTACCGGGCGTATTTTTTGAGTTGTCGAGATTTTCAGGAGCTAAGGA
AGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATC
GTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTT
CAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCC
GGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTACGTATGGCAA
TGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCAT
GAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTT
TCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTA
AAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGT
TTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAA
ATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCG
TTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAG
TGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGGTTG
CTACGCCTGAATAAGTGATAATAAGCGGATGAATGGCAGAAATTCGGATCTCGACCGCG
TTTGGGCGGTGGCTCCCTGCCACGCGGCTCCGAACAGAAGCTGATCTCCGAAGAGGATC
TGATTACCCTGTTATCCCTACCCTAAAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAA
GGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGC
CTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGG
TGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGC
AGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAG
ATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCA
GCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGC
TCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTC
TGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCT
TTCGACCTGCAGCCCAAGCTTACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCAC
CCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCG
CCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAA
CTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGC
CGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGA
TCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAA
GGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGT
CTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGG
CGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCC
TTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCG
CACCTGGTGCATGACCCGCAAGCCCGGTGCCTGACGCCCGCCCCACGACCCGCAGCGCC
CGACCGAAAGGAGCGCACGACCCCATGCATCGATAAAATAAAAGATTTTATTTAGTCTC
CAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGAGAACCATCA
GATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCA
ATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGC
CCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGT
GTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG
GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATGGGTAACAGTTTCT
TGAAGTTGGAGAACAACATTCTGAGGGTAGGAGTCGAATCGAGAGAGAGAGAGAGAGAG
AGAGAGAGAGAGAGAGAGAGAGAGACGTGGGCCCAATTCTGTCAGCCGTTAAGTGTTCC
TGTGTCACTGAAAATTGCTTTGAGAGGCTCTAAGGGCTTCTCAGTGCGTTACATCCCTG
GCTTGTTGTCCACAACCGTTAAACCTTAAAAGCTTTAAAAGCCTTATATATTCTTTTTT
TTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGATTTATATTAATTTTAT
TGTTCAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTTAGCCATGAGA
GCTTAGTACGTTAGCCATGAGGGTTTAGTTCGTTAAACATGAGAGCTTAGTACGTTAAA
CATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTACTATCAACAGGTTGAACT
GCTGATCAACAGATCCTCTACACTAGAAGGGACGCACCGCTAGCAGCGCCCCTAGCGGT
ATCCTATAAAAAAACACACCGCGCCGCTAGCAGCACCCCTAATATAAAATAATGTTTTT
TATAAAAATAGTCAGTACCACCCCTACAAAACGGTGTCGGCGCGTTGTTGTAGCCGCGC
CGACACCGCTTTTTTAAATATCATAAAGAGAGTAAGAGAAACTAATTTTTCATAACACT
CTATTTATAAAGAAAAATCAGCAAAAACTTGTTTTTGCGTGGGGTGTGGTGCTTTTGGT
GGTGAGAACCACCAACCTGTTGAGCCTTTTTGTGGAGTGGGTTAAATTATACTAGCGCG
TTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGC
GCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCG
CCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCAC
ACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCG
GCAAGCAGGCATCGCCATGTGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTG
AGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTG
ATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGT
GGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATG
ATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTC
GCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAG
GAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGG
GCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAA
CACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCT
CCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGAT
CCTCATCCTGTCTCTTGATCAGATCT
pSHAG-MAGIC vectors must be
propogated in PIR1 E.coli. These are
available from Invitrogen (product #C101010) or, alternately, from Steve Elledge’s lab
(selledge@genetics.med.harvard.edu).